TYROSINASE-ACTIVATOR PROTEIN FUSION ENZYME

Abstract: The present invention relates to a nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein. Activator proteins used in the present invention are ORF438 and URF402. Prokaryotic tyrosinases that require a separate activator protein, particularly tyrosinases derived from <u>Streptomyces</u> are preferred. It is also preferred that the activator protein sequence be positioned (5') relative to the tyrosinase sequence. The present invention also relates to a fusion enzyme which comprises an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein, which may also contain a linker positioned between the amino acid sequences of the activator protein and the tyrosinase. The present invention also relates to melanin produced by a fusion enzyme of the present invention.

Description: TITLE OF THE INVENTION TYROSINASE-ACTIVATOR PROTEIN FUSION ENZYME This application is a continuation in part of application Serial No. 7/857,602, filed March 30, 1992. This application is also a continuation-in- part of application serial No. 923,692 filed July 31, 1992 which is a continuation-in-part of applications Serial No. 600,244, filed October 22, 1990, Serial No. 641,617, filed January 16, 1991, and Serial No. 737,899 filed July 26, 1991. Serial No. 600,244 is a continuation of application Serial No. 310,881, filed February 17, 1989, now abandoned, which is a continuation-in-part of applications Serial No. 160,766 and 160,771, both filed on February 26, 1988 and now abandoned. Serial No. 641,617 is a continuation of application Serial No. 347,637, filed May 5, 1989, now abandoned. Serial No. 737,899 is a continuation of application Serial No. 363,138, filed June 8, 1989, now abandoned, which is a continuation- in-part of application Serial No. 219,279, filed July 15, 1988 and now abandoned. The disclosures of all of__the foregoing are incorporated by reference. BACKGROUND OF INVENTION Melanogenesis, production of the biological polymer melanin, is a widespread phenomena in nature occurring in most phyla from fungi to mammals. The black, brown, buff and Tyndall-blue pigments found in feathers, hairs, eyes, insect cuticle, fruit and seeds are usually melanins. Melanins have been assigned a photoprotective role in the skin, their role in the eye and inner ear is unknown. Melanins are also found in the mammalian brain where they are referred to as neuromelanins. The biological function of neuromelanins is unknown. The stepwise biosynthesis of melanins is depicted in Figure 1. Tyrosinase (E.C. 1.14.18.1) catalyzes two types of reactions involved in the biosynthesis of melanin: the orthohydroxylation of monophenols to catechols, which is referred to as cresolase activity, and the dehydrogenation of catechols to o-quinones, designated as catecholase activity. Molecular oxygen is used for the hydroxylation reaction. For this reason, tyrosinase acting on a monophenol is referred to as a "mixed function oxidase" . Hayaishi, in "Biological Oxidation" (Singer, ed.) p.581, Interscience Publishers, New York (1968) . As used herein tyrosinase refers to all enzymes possessing the above described enzymatic activity. Tyrosinases are also sometimes referred to as polyphenol oxidases. As can be seen from the biosynthetic pathway of melanin depicted in Figure 1, tyrosinase is essential to the production of melanin. Therefore, the ability of an organism to express significant quantities of tyrosinase activity is essential to the organism's ijn vivo production of melanin. Tyrosinase is not naturally found in all organisms. Rather, tyrosinase has been found to occur in a relatively limited number of prokaryotes, is absent in a variety of higher plants and is generally confined to specific cells of the skin in higher animals but may also occur in interior tissue, such as the substantia nigra, eye and inner ear. Given the limited number of organisms that produce melanin, one objective of the present invention is to provide a means for introducing tyrosinase activity into organisms that are otherwise unable to produce melanin. Even in those organisms where tyrosinase activity occurs naturally, such activity is generally present at low levels. As a result, melanin is generally produced in small quantities by those organisms that possess tyrosinase activity. It is therefore a further objective of the present invention to provide a means for enhancing the level of tyrosinase activity in organisms in order to enhance the in vivo production of melanin. Several prior art references teach how to genetically engineer an organism to possess tyrosinase activity. In U.S. Application Serial No. 7/857,602, filed March 30, 1993, which is incorporated herein by reference, Applicants teach a method for producing melanin from transformed microorganisms wherein a sequence encoding for tyrosinase is introduced into the organism. Microorganisms that have be genetically engineered to enhance their abilities to produce tyrosinases include, but are not limited to species of Streptomvces. Escherichia. Bacillus. Salmonella. Staphvlococcus. and Vibrio. For example, cloned tyrosinase genes from Steptomyces sp. have been shown to produce melanin pigments in culture. J. Gen. Microbiol. 129:2703-2714 (1983) ; QSΩS. 21:101- 110 (1985) . The cloned genes have also been expressed in Streptomvces and E. coli. della-Cioppa, Bio/Technology £:634-638 (1990). In each case, both tyrosinase and ORF438 were required for melanin production. U.S. Patent No. 4,898,814 issued to Kwon discloses a cDNA clone of human tyrosinase and claims a method of making human tyrosinase by expressing the cDNA in 1. coli. Many forms of tyrosinase from bacteria, such as Streptomvces. require an activator protein. For example, the el locus of S. antibioticus has been shown to contain two open reading frames (ORF's) that encode a putative ORF438 protein (M r =14,754) and tyrosinase (M r =30,612) . ORF438 and tyrosinase are thought to be transcribed from the same promoter in S. antibioticus. Bernan, et al. , Gene 12:101 (1985) . Both genes are required for melanin production. Bernan, et al. , Gene 3_7:101 (1985). Based on genetic evidence, ORF438 protein has been shown to function as a trans-activator of tyrosinase. Lee, et al., Gene £5:71 (1988) . It has been suggested that the ORF438 protein is involved in tyrosinase secretion, or it may function as a metallothionein-like protein that delivers copper to tyrosinase, Bernan, et al. , Gene 37:101 (1985); Lee, et al. , Gene 65:71 (1988) . The mel locus of S. glaucescens has a nearly identical ORF sequence upstream of tyrosinase that probably serves a similar function. Huber, et al. Biochemistry 2 =6038 (1985); Huber, et al. , Nucleic Acids Res. 1^:8106 (1987) . The existence of an ORF438 protein, however, has never been confirmed in vivo. The melanin operons of S. antibioticus and S. glaucescens have been isolated and sequenced, and both share sequence homology and similar gene arrangement. The polypeptide sequence encoded by 0RF438 (146 amino acids) in S. antibioticus is structurally and functionally equivalent to URF402 (134 amino acids) from S. glaucesecens Huber, et al. Nucleic Acids Res. 13. -8106 (1987) . Disruption of the URF402 coding sequence abolishes the melanin phenotype similar to that already known for ORF438. Recent evidence suggest that the ORF438 protein (and by analogy the URF402 protein equivalent) functions as a molecular chaperone for tyrosinase. Chen, et al., J. Biol Chem. 268:18710 (1993). Tyrosinase has also been isolated and employed to produce melanin in vitro. For example, in U.S. Application Serial No. 7/982,095, filed November 25, 1992, which is incorporated herein by reference, Applicants teach the production of melanin in vitro using a tyrosinase which is excreted from the microorganism during fermentation. In vitro production of melanin is dependant on the production of significant quantities of tyrosinase activity. Tyrosinase activity may be lost during isolation of the secreted tyrosinase due to a disruption of the tyrosinase - activator protein complex. It is therefore an objective of the present invention to stabilize the tyrosinase - activator protein complex in order to reduce tyrosinase activity loss during isolation and purification. Fusion proteins have been synthesized to overcome instability and proteolytic degradation of a polypeptide of interest. Many eucaryotic proteins have been produced in E. coli as fusion proteins with E. coli polypeptides such as Beta-galactosidase. Beta-galactosidase can be used to protect the foreign passenger protein from degradation in E. coli. Somatostatin, the first eurcaryotic protein to be produced in E. coli was produced by fusing a synthetic gene to the entire Beta-galactosidase coding sequence (Itakura, et al., Science 198:1056 (1977)) and somatostatin was subsequently isolated by chemical cleavage of the fusion protein. When two independently funtioning polypeptides are fused into a single polyprotein by way of a synthetic gene fusion, the resulting gene construct can be engineered for expression behind a single promoter, ribosome binding site, and initiation codon. A gene fusion constructed in such a way can be placed in a single location within a chromosome or episomal element for subsequent expression of the fusion protein. Such fusion genes are inherently more stable because both gene sequences are coordinately expressed at high levels from a single promoter. Undesirable effects such as differential down regulation or rearrangement and deletion of one of the genes can thus be avoided. Unfortunately, however, most fusion protein constructs are not functional. Proteins fold into conformationally active states as dictated by their primary amino acid sequence. Additional polypeptide sequences at the C- or N- terminus can interfere with proper folding, thereby preventing the formation of a biologically active protein. Enzymes typically fold into well defined three-dimensional conformations to allow the formation of a catalytic pocket that excludes potential substrate molecules of abnormal size or conformation, but allows access of substrate molecules with the correct three-dimensional structure. Many enzymes will not function as fusion polypeptides because the sequence extensions interfere with proper folding, or interfere by sterically blocking the substrate's access to the catalytic site. The present invention relates to Applicants' recognition that it might be possible to construct a biologically active fusion enzyme coupling tyrosinase with an activator protein. It has been shown that histidine residues #102 and # 117 of the ORF438 activator protein are critical for copper binding and delivery to the active site of tyrosinase. Chen, et al., J. Biol. Che . 2£&:18710 (1993). In order to form a biologically active fusion enzyme between tyrosinase and an activator protein, both functional domains of tyrosinase and the activator protein must be correctly folded. Further, the fusion enzyme must be able to assume a structural conformation enabling the activator protein to intermolecularly deliver copper to the active site of the tyrosinase to form a biologically active tyrosinase. Based on the existing prior art, it is unclear whether such a fusion enzyme would be biologically active. SUMMARY OF INVENTION The present invention relates to a nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein. Activator proteins used in the present invention include but are not limited to ORF438 and URF402. Tyrosinases used in the present invention include both prokaryotic and eucaryotic tyrosinases. Prokaryotic tyrosinases that require a separate activator protein, particularly tyrosinases derived from Streptomvces are preferred. It is also preferred that the activator protein sequence be positioned 5' relative to the tyrosinase sequence. The present invention also relates to a vector useful for introducing a nucleic acid sequence encoding a fusion enzyme into an organism. The vector comprises a nucleic acid sequence encoding for a fusion enzyme of the present invention and a promoter sequence that regulates the transcription of fusion enzyme. The present invention also relates to organisms, such as bacteria, yeast fungi, plants and animals which have been transformed by the vector of the present invention. The present invention also relates a fusion enzyme which comprises an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein. The fusion enzyme may also contain a linker positioned between the amino acid sequences of the activator protein and the tyrosinase. The present invention also relates to melanin produced by a fusion enzyme of the present invention. The melanin may be produced in vi tro by contacting a fusion enzyme with an enzyme substrate under suitable reaction conditions to form melanin. Melanin may also be produced in vivo by an organism transformed with a vector of the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1 depicts the stepwise biosynthesis of melanins. Figure 2 provides the plasmid map of pBGC623. Figure 3 provides the plasmid map of pBGC635. Figure 4 provides the plasmid map of pBGC636. Figure 5 provides the plasmid map of pBGC646. Figure 6 provides the plasmid map of pBGC648. Figure 7 provides a comparison of the melanin production capabilities of E. coli when transformed by plasmids pBGC623, pBGC635, pBGC636, pBGC646 and pBGC648. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a nucleic acid sequence encoding for a fusion enzyme of tyrosinase and an activator protein for tyrosinase. The present invention also relates to a vector useful for transforming a host organism that contains the nucleic acid sequence of the present invention. The present invention also relates to the expression of a fusion enzyme of a tyrosinase and an activator protein by the transformed organism. Host organisms that may be transformed to express the fusion enzyme of the present invention include but are not limited to bacteria, yeast fungi, plants and animals. Expression of the fusion enzyme by the host organism enables and/or enhances tyrosinase activity in the host thereby enabling and/or enhancing melanin production by the host. The present invention also relates to a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for an activator protein of tyrosinase. Finally, the present invention relates to the in vivo and in vitro production of melanin using the fusion enzyme of the present invention. The fusion enzyme of the present invention comprises an amino acid sequence for a tyrosinase and an amino acid sequence for an activator protein. The nucleic acid sequences for both proteins are under the same promoter control and are thus expressed as a single peptide. Tyrosinases used in the present invention include both prokaryotic and eucaryotic tyrosinases. In some organisms, tyrosinases are referred to as polyphenol oxidases. Tyrosinases from prokaryotes are preferred, particularly those tyrosinases that require a separate activator protein such as those that have been obtained from Streptomyces. Activator proteins used in the present invention include but are not limited to ORF438 and URF402. The activator protein employed in the present invention need not naturally occur in the organism from which the tyrosinase is derived. Rather, activator proteins from a variety of sources should function with a given tyrosinase in view of the similarity of the copper binding sites of different tyrosinases. See Chen, et al., J. Biol. Chem. 268 18710 (1993) . The fusion enzymes of the present invention are preferably constructed such that the amino acid sequence for the activator protein is positioned on the N- terminus of the tyrosinase amino acid sequence. As can be seen from Example 5, fusion enzymes where the activator protein is positioned on the N- terminus of tyrosinase exhibit equivalent melanin production capabilities as where tyrosinase and the activator protein are expressed separately. Eucaryotic tyrosinases tend to comprise higher molecular weight (50-70 kD) single polypeptide chains without defined activator proteins. Applicants speculate that the activator equivalent of the ORF438 protein is built into the polypeptide backbone of eucaryotic tyrosinases. Based on the amino acid homology at the active sites between eucaryotic and Streptomyces tyrosinases, it appears that excess polypeptide sequences occur in the C- terminal region of the larger eucaryotic enzymes. Based on this observation, it might be predicted that ORF438 would function best if fused to the C- terminus rather than the N- terminus of the 30 kD Streptomyces tyrosinase since the fusion protein would then mimic the eucaryotic tyrosinases with respect tp placement of the catalytic site. However, since many newly synthesized polypeptides undergo three dimensional folding into their preferred active conformation beginning with the free C- terminus, it might also be expected that C- terminal additions are disruptive to proper folding and, hence, normal catalytic activity. Hence, prior to preparing the fusion enzymes of the present invention, it was unclear whether a fusion enzyme at either the N- or C- terminus would be functional. The amino acid sequence encoding the activator protein need not be directly attached to the tyrosinase sequence. In plasmid pBGC648, a His residue has been inserted between the activator protein and the tyrosinase sequence. A single amino acid and a repeating Pro-Thr amino acid sequence, which behaves as a natural hinge, are preferred as linking sequences between tyrosinase and the activator protein. Determination of the wide variety of amino acid sequences that may be interposed between the tyrosinase and activator protein sequences can be routinely determined by one of ordinary skill in the art in view of the teachings of the present invention. Other polypeptide linkers that are known from the literature to provide a high degree of flexibility between two polypeptide sub-domains might work equally as well as linking sequences between the tyrosinase sequence and the activator protein sequence. One example of this type of hinge polypeptide is a proline - threonine repeating unit such as those known form cellulose binding proteins. Ong, et al., Bio/Technology 7 604(1989). Construction of synthetic oligonucleotide linkers that encode hinge polypeptides of known flexibility is within the level of ordinary skill. In addition, synthetic oligonucleotide linkers could be used to construct random polypeptide linker sequences to join the tyrosinase - activator protein domains together. Linkers with a high degree of flexibility and sufficient length might be expected to work best for intermolecular cis-activation of tyrosinase. Inflexible linker polypeptides, or extremely short linker polypeptides, might be expected to permit only intramolecular trans-activation of neighboring fusion enzymes. In order to provide a clear and consistent understanding of the specification and the claims, including the scope given to such terms, the following definitions are provided: Activator protein: a gene product that alters, activates or enhances the activity of tyrosinase. The activator protein may function as a trans-activator, as a metallothionein-like protein that delivers an ion to a tyrosinase apoenzyme or it may function in assisting the secretion of tyrosinase. ORF438 gene, URF402 and ORF(s) 3' to the tyrosinase coding sequence code for activator proteins that enhance melanogenesis in all of the ways described. Melanin: Melanins are polymers produced by polymerization of reactive intermediates. The polymerization mechanisms include but are not limited to autoxidation, enzyme catalyzed oxidation and free radical initiated polymerization. The reactive intermediates are produced chemically or enzymatically from precursors. Suitable enzymes include, but are not limited to peroxidase and catalases, polyphenol oxidases, tyrosinases, tyrosine hydroxylases or laccases. The precursors which are converted to the reactive intermediates are hydroxylated aromatic compounds. Suitable hydroxylated aromatic compounds include, but are not limited to 1) phenols, polyphenols, aminophenols and thiophenols of aromatic or polycyclic aromatic hydrocarbons, including but not limited to phenol, tyrosine, pyrogallol, 3- aminotyrosine, thiophenol and a-naphthol; 2) phenols, polyphenols, aminophenols, and thiophenols of aromatic heterocyclic or heteropolycyclic hydrocarbons such as but not limited to 2-hydroxypyrrole, 4-hydroxy- 1,2-pyrazole, 4-hydroxypyridine, 8- hydroxyquinoline, and 4,5- dihydroxybenzothiazole. Suitable hydroxylated aromatic compounds also include X/L-tyrosine, L-tyrosine/X and X/L- tyrosine/X where X is a single amino acid, a dipeptide or an oligopeptide bound to L- tyrosine. The nucleic acid sequences encoding for the fusion enzymes of the present invention may be inserted into a wide variety of vector constructs known in the art for transforming a host organism. Suitable techniques include those described in Maniatis, et al. , Molecular Cloning. 1st Ed., Cold Spring Harbor Laboratory, New York (1982) ; Molecular Cloning. 2nd Ed., Cold Spring Harbor Laboratory, New York (1989) ; Methods in Enzvmology. Vols. 68 (1979), 100 (1983), 101 (1983), 118 (1986) and Vols. 152-154 (1987) DNA Cloning. Glover, Ed., IRL Press, Oxford (1985); and Plant Molecular Biology: Manual. Gelvin, et al., Eds., Kluwer Academic Publishers, Podrecht (1988) . Medium compositions have been described in Miller, Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, New York (1972) , as well as the references previously identified. Hopewood, et al., "Genetic Manipulation of Streptomyces: A Laboratory Manual", The John Innes Foundation, Norwich, England (1985) . With regard to the expression of the tyrosinase- activator protein fusion enzyme of the present invention in a transformed microorganism, it is preferred that the transformed organism be grown under the conditions described in U.S. Application Serial No. 857,602, filed March 30, 1992 which is incorporated herein by reference. With regard to the expression of the tyrosinase- activator protein fusion enzyme of the present invention in plants, it is preferred that the nucleic acid cassette encoding the fusion enzyme be inserted into one of the viral constructs described in U.S. Application Serial No. 923,692, filed July 31, 1992, or U.S. Application Serial No. 997,733, filed December 30, 1992, which are both incorporated herein by reference. Vectors encoding the tyrosinase-activator protein fusion enzyme of the present invention may be produced by standard techniques. Appropriate vectors which can be utilized as starting materials are known in the art. The DNA sequence coding for the fusion enzyme is inserted into an appropriate vector in such a manner that the enzyme is correctly expressed. In other words, the DNA sequence is positioned in the proper orientation and reading frame so that the correct amino acid sequence is produced upon expression of the DNA sequence in the host. In accordance with conventional techniques, a chimeric DNA sequence is generally constructed which contains a promoter operable in the specific host and the DNA sequence coding for the desired enzyme. The chimeric DNA sequence may further contain 3' non-coding sequences operable in the host. The chimeric DNA sequence can be prepared in situ within a suitable vector by inserting the DNA sequence coding for the enzyme into a restriction site of a known host transformation vector. Alternatively, the chimeric gene could be first constructed and then inserted into a vector to produce a transformation vector. The vector can be further modified by utilizing an enhancer sequence and/or a strong promoter, which leads to an increased production of the fusion enzyme. The typical vector is a plasmid having one or more marker genes for antibiotic resistance, an origin of replication, and a variety of useful restriction sites for cloning or subcloning restriction fragments. A large number of vectors have been described which are useful for transforming many microorganisms including but not limited to Streptomyces and £. coli. See, for example, Cloning Vectors. Pouwels, et al. ed. Elsevier Science Publishers Amsterdam (1985) . A large number of naturally occurring Streptomyces plasmids have been described, many of which are conjugally proficient. Two such isolates, SLP1.2 and pIJlOl, have formed the basis of a series of useful plasmid vectors. Thompson, et al. , " Gene 2£:51 (1982) . The plasmids of the SLP1 family, of which SLP1.2 is the largest detected member, were discovered as autonomous replicons in S. lividans 66 after interspecific matings with S. coelicolor A3 (2) . The SLP1 replicon is integrated in the S. coelicolor genome but can be excised together with various lengths of neighboring DNA to become autonomous in S. lividans. The SLP1 plasmids exist stably at a copy number of 4-5 per chromosome in S. lividans and have a narrow host range. The 8.9 kb plasmid pIJlOl was discovered in £_-. ljvjdan-g ISP5434 (Kieser, et al. , Mol. Gen. Genet. l£jL:223 (1982)) but can be conjugally transferred to a wide variety of Streptomvces species. Derivatives (e.g. pIJ102) have been isolated from the plasmid which have similar properties but are smaller. Kieser, et al. (1982), supra. Plasmid pIJlOl has a copy number of 100-300 per chromosome equivalent in most hosts and a minimum replicon of less than 2.1 kb. Derivatives carrying drug-resistance determinants have been constructed to act as vectors, and a chimeric plasmid which can be used as a shuttle vector between £. coli and Streptomyces is available. The temperate phage C31 has a wide host range within the Streptomycetes and lysogenizes S. coelicolor A-3(2) via a site-specific integration event. Lomovshaya, et al. , Bacteriol Rev. 44. 206 (1980) . Up to 42.4 kb of DNA can be packaged within a viable phage particle, but only 32 kb (at the most) of the DNA contains the genetic information essential for plaque formation. Derivatives of φC31 containing deletions can be used as vectors, and recombinant phages can either be grown lytically or used to lysogenize suitable streptomyces strains. pBR322-derived plasmids are very common for use in £. coli transformation. They possess a pair of antibiotic resistance genes which confer antibiotic resistance when Escherichia coli are successfully transformed. Typically, the insertion of a DNA segment is made so that one of the antibiotic resistance genes is inactivated. Selection then is accomplished by selecting for fi. coli exhibiting antibiotic resistance conferred by the second gene. Bolivar, et al., Gene 2:95 (1977); and Sutcliff, J.. Proc. Natl. Acad. Sci .. USA 75:3737 (1978) . Another example of transforming vectors is the bacteriophage. The M13 series are modified filamentous E. coli bacteriophage containing single stranded circular DNA. The M13 series carry the lacZ gene for β-galactosidase and will metabolize the galactose analog Xgal to produce a blue color. Placing a cloned insert into the polylinker sequence located in the amino terminus of the lacZ gene inactivates the gene. Microorganisms carrying an M13 with an inactivated lacZ (representing a cloned insert) are distinguishable from those carrying an M13 with an active lacZ gene by their lack of blue color. Messing, et al. , Proc. Natl. Acad. Sci.. USA 4:3642 (1977); and Messing, Methods in Enzymology 101:20 (1983) . Other transforming vectors are the pUC series of plasmids. They contain the ampicillin resistance gene and origin of replication from pBR322, and a portion of the lacZ gene of fi. £aϋ. The lac region contains a polylinker sequence of restriction endonuclease recognition sites identical to those in the M13 series. The pUC series have the advantage that they can be amplified by chloramphenicol. When a DNA fragment is cloned into the lac region the lac gene is inactivated. When £. ςoli containing a pUC plasmid with an inactivated lacZ gene is grown in the presence of isopropylthiogalactoside (IPTG) and 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside (Xgal) its colonies are white. If it carries a pUC plasmid with an active lacZ gene its colonies are blue. Vieira, et al. , Gene 19:259 (1982). Bacteria are transformed by means conventional in the art. The genus Streptomyces is one of three aerobic genera of bacteria of the order Actinomycetales. Streptomyces are Gram-positive, mycelial, spore- forming bacteria. Several naturally occurring Streptomyces plasmids have been described. Streptomyces lividans TK64 has no tyrosinase gene and produces no melanin. Applicants teach the transformation of Streptomyces lividans TK64 with plasmid pIJ702 which encodes for the tyrosinase gene in U.S. Application Serial No. 7/857,602, filed March 30, 1992 which is incorporated herein by reference. Transformation is carried out by means standard in the art. Similarly, transformation of Streptomyces can be performed using a plasmid encoding the fusion enzyme of the present invention. Transformation vectors of the present invention may also be used to transform a variety of microorganisms after insertion into vectors which are useful for transforming the corresponding host microorganism. Bluescript (obtained from Stratagene, LaJolla, CA) is a pUC derivative having a β-galactosidase color indicator and a 3-ac promoter. In U.S. Application Serial No. 7/857,602, Applicants teach modification of the Bluescript plasmid by inserting a tyrosinase gene. This modified plasmid was used to successfully transform £. coli which formed pigmented colonies. Similarly, the Bluescript may be modified by inserting a nucleic acid sequence encoding for the fusion enzyme of the present invention. Melanin has been purified from bacterial cells with 0.5N NaOH at room temperature and at 100°C. Pigmented fractions were found to be: (1) soluble in acid and base; (2) soluble in ethyl alcohol and base; and (3) soluble base only. Pavlenko, et al. , Microbiology USSR 5_fi 539 (1981) Soluble melanin can be extracted from the medium and purified. This is done by first removing cells and particulate matter using, for example, filtration or centrifugation. A variety of filtration methods are known in the art including filtration through glass wool. If centrifugation is used, 5,000 X gravity is usually sufficient. The melanin is then precipitated at between pH 2-4, preferably about 3. Precipitated melanin is removed by either filtration or centrifugation. The melanin is washed by successive resolubilization at high pH, i.e. about pH 7.0 to about pH 9.0, preferably about pH 8.0, and precipitation at low pH followed by filtration or centrifugation. The melanin may also be concentrated using molecular weight filtration, such as reverse osmosis. Salt precipitation can be as effective in precipitating the melanin as low pH. The invention is further illustrated by the following non-limiting examples. EXAMPLES 1. Preparation of Plasmids PBGC623. PBGC635 and PSGC636 " Plasmid pBGC623 is a plasmid containing the nucleic acid sequence for ORF438. Figure 2 provides the plasmid map of pBGC623. Plasmid pBGC635 is a plasmid containing a nucleic acid sequence for tyrosinase. Figure 3 provides the plasmid map of pBGC635. Plasmid pBGC636 is a plasmid encoding for both ORF438 and tyrosinase under separate promoters. Figure 4 provides the plasmid map of pBGC636. Preparation of plasmids pBGC623, pBGC635 and pBGC636 are taught in U.S. Application Serial No. 7/857,602 which is incorporated herein by reference. 2. Preparation <?f B(3C$4$ In order to prepare pBGC646, the Bel I site at the stop codon of the tyrosinase gene (in pBGC188Nde) was blunted with mung bean nuclease and ligated with a synthetic Eco RI linker (dGGAATTCC; SEQ. NO. 1) . The tyrosinase gene was removed from plasmid pBGC188Nde as a 822bp Nde I/Eco RI fragment and gel purified in low melt agarose. The purified fragment was ligated into an ORF438 containing plasmid (pBGC623) that was modified as follows. Plasmid pBGC623 was cleaved with Nco I, blunted with mung bean nuclease, and ligated with a synthetic Eco RI linker (dGGAATTCC) . The plasmid was gel purified in low melt agarose and ligated with the Nde I/Eco RI fragment containing the modified tyrosinase gene. Figure 5 provides the plasmid map of pBGC646 [SEQ. NO. 2] . The translated amino acid sequence for the fusion enzyme encoded for by plasmid pBGC646 is provided as SEQ. NO. 3. Transfor ants were screened in HB101 and two independent transformants were identified as having the correct orientation. These plasmids are pBS646.9 and pBS646.19, and both are identical. Both plasmids were transformed into E. coli strain K38 that harbors plasmid pGPl-2 and plated on agar containing tyrosine and copper. Both transformants gave a melanin phenotype as described previously (della, Cioppa, et al. Bio/Technology £:634-638 (1990)), and were shown by SDS-PAGE to give a ~45kD band by SDS-PAGE. The hybrid tyrosinase/ORF438 fusion enzyme is predicted to encode a 45,806 MW protein with four additional amino acids linking the two functional domains (NH2- tyrosinase-trp-asn-ser-ala-ORF438-COOH) . 3. Preparation of PBGC648 To construct a second hybrid fusion gene between ORF438 and tyrosinase, the two synthetic oligonucleotides shown below S'-dCCAGGGCGC^CGGCTCCTCOCCTTCCCCTCCAACCA-S' [SEQ. NO. 41 a'-dTCGAGGTCCCGCGGGCCGAGGAGGGGAAGGGGAGGTTGGTAT-S' [SEQ. NO. 5) were annealed, kinased, and used to clone into the Sac I site of the ORF438 gene. This replacement oligonucleotide sequence results in the destruction of the TGA stop codon of ORF438 and creates an in- frame Ndel site in its place for insertion of tyrosinase. A triple ligation reaction was set up that included the annealed oligonucleotide shown above, a 2,934 bp Hind Ill/Sac I fragment form pBGC623, and a 1,107 bp Ndel/Hind III fragment from pBGC635. The new plasmid (pBGC648) creates an ORF438/tyrosinase in-frame gene fusion that encodes a single polypeptide chain of 46,230 Daltons. The introduction of the new Ndel site introduces a single histidine residue between the two coding sequences upon translation. Figure 6 provides the plasmid map of pBGC648. The nucleic acid sequence for pBGC648 is provided as SEQ. NO. 6. The translated amino acid sequence for the fusion enzyme encoded for by plasmid pBGC648 is provided as SEQ. NO. 7. The DNA at the junction of the ORF438 and tyrosinase genes in plasmid pBGC648 were sequenced. Approximately 180 bp 3' of the Sac I site in the fusion were found to be correct, and the translated amino acid sequence is as predicted (See SEQ. No. 7) . Plasmid pBGC648 was transformed into E. coli strain K38 that harbors plasmid pGPl-2 and plated on agar containing tyrosine and copper. The transformants gave a black melanin phenotype as described previously (della-Cioppa, et al., Bio/Technology 1:634-638 (1990)), and were shown by SDS-PAGE to give a -46kD band by SDS-PAGE. Transformants harboring pBGC648 give rise to the black melanin phenotype as rapidly as that seen when ORF438 and tyrosinase are expressed as single polypeptide chains from single genes. Phenotypically, the ORF438/tyrosinase gene fusion retains full catalytic activity similar to the wild type -30kD tyrosinase holoenzyme. 4. Transformation of E. coli with pBGC623. pBGC635. P BGC636. PBGC646 and PBGC648 Plasmids pBGC623, pBGC635, pBGC636, pBGC646 and pBGC648 were introduced into E. coli by pretreating exponentially growing cultures of the E. coli with CaCl 2 as described in Maniatis, et al. , Molecular Cloning (1982) . 5. Comparison of Melanin Production By E. coli transformed with PBGC623. PBGC635, PBGC636. pBGC646 and PBGC648 Plasmids pBGC623, pBGC635, pBGC636, pBGC646 and pBGC648 were each introduced into E. coli. The transformed strains of E. coli were then grown on agar plates containing tyrosine and copper as described previously in della-Cioppa, et al. , Bio/Technology £ 634 (1990) in order to evaluate each strains ability to produce melanin. Figure 7 provides a comparison of the melanin production capabilities of E. coli when transformed by these five different plasmids. The amount of melanin formed was determined by the size and color intensities of black melanin halos that formed around the transformed E. coli colonies. The rate of color development on agar plates, and the intensity of the black halo formation, is directly proportional to the level of tyrosinase enzymatic activity in each of the different plasnid bearing E. coli colonies. Melanin formation was quantitated as (-) none, (+) very weak, (++) weak, (+++) moderate, (++++) strong and (+++++) very strong. E. coli transformed with either pBGC623 or pBGC635 do not produce a positive black melanin phenotype. E. coli transformed with pBGC636 produced a positive black melanin phenotype. E. coli transformed with pBGC646 gave rise to a positive black melanin phenotype. However, E. coli transformed with pBGC646 produced melanin at a slower rate than E. coli transformed with pBGC636 in which ORF438 and tyrosinase are expressed as single polypeptide chains from single genes. E. coli transformed with pBGC648 produced a positive black melanin phenotype at a comparable rate as pBGC636, indicating that the fusion enzyme produced by pBGC648 functions equally well as when ORF438 and tyrosinase are expressed independently. 6. Expression Of Tyrosinase/ORF4 8 Fusion Bnzvme Containing Mprppiast Targeting Sequence in Plants For expression of a tyrosinase - activator protein fusion enzyme in higher plants, it may be advantageous to target the fusion enzyme to the chloroplast. Chloroplasts are known to contain the enzymatic pathway for production of L-tyrosine (the primary substrate for tyrosinase) , and the oxidative environment inside the chloroplast may be well suited for achieving optimal enzymatic activity. In order to target a tyrosinase - activator protein fusion enzyme to chloroplasts, the nucleotide sequence encoding the chloroplast transit peptide (CPT) from ribulose bisphosphate carboxylase small subunit (RuBPCase SSU) from Nicotiana tabacum was cloned (as an Nco I/Sph I fragment) and fused by way of its naturally occurring Sph I site (at the cys- met cleavage site of the CTP) to the naturally occurring Sph I site at the N-terminus of ORF438. The CTP/ORF438 nucleotide fusion was then exchanged as an Nco I/Sac I fragment in the plasmid BlueScript™ that contained the 0RF438/tyrosinase sequence as described in plasmid pBS648. The resulting nucleotide sequence and translated amino acid sequence of the CTP/ORF438/tyrosinase fusion are shown in SEQ. No. 8 and SEQ. No. 9 respectively. The CTP/ORF438 tyrosinase fusion gene encodes 478 amino acids residues of which the 57 at the N-terminus direct the ORF438/tyrosinase fusion into the chloroplast. Upon import into the chloroplast compartment, the 57 amino acid CTP is proteolytically removed thus resulting in the identical ORF438/tyrosinase fusion enzyme of -45 kD as previously produced in E. Coli. The CTP/ORF438/tyrosinase fusion enzyme has a deduced molecular weight of 51,461 Daltons. The CTP/ORF438/tyrosinase nucleotide sequence may then be inserted into a viral construct such as those described in U.S. Application Serial No. 923,692, filed July 31, 1992, or U.S. Application Serial No. 997,733, filed December 30, 1992 and used to systemically infect higher plants. While the invention has been disclosed by reference to the details of preferred embodiments, the disclosure is intended in an illustrative rather than in a limiting sense, as it is contemplated that modifications will readily occur to those skilled in the art, within the spirit of the invention and the scope of the appended claims. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Della-Cioppa, Guy Kumagai, Monto (ii) TITLE OF INVENTION: TYROSINASE-ACTIVATOR PROTEIN FUSION ENZYME (ϋi) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Pennie & Edmonds (B) STREET: 2730 Sand Hill Road (C) CITY: Menlo Park (D) STATE: California (E) COUNTRY: U.S.A. (F) ZIP: 94025 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE:Patent in Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US 08/152,483 (B) FILING DATE: November 12, 1993 (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 7/857,602 (B) FILING DATE: March 30, 1992 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 923,692 (B) FILING DATE: July 31, 1992 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 600,244 (B) FILING DATE: October 22, 1990 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 641,617 (B) FILING DATE: January 16, 1991 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 737,899 (B) FILING DATE: July 26, 1991 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Halluin, Albert P. (B) REGISTRATION NUMBER: 25,227 (C) REFERENCE/DOCKET NUMBER: BIOG-20240/8129-040 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 854-3660 (B) TELEFAX: (415) 854-3694 (C) TELEX: 66141 PENNIE (2 ) INFORMATION FOR SEQ ID NO : 1 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 8 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GGAATTCC 8 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4294 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI r SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT60 GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC120 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CAGTGCTGCA ATGATACCGC180 GAGACCCACG CTGACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG240 AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG300 AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG360 GCATCGTGGT GTCACGCTCG GCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT 20 CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC480 CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC540 ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA600 CCAAGTATTT GGAAGATGCG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA660 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA720 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA780 ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC840 AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC900 TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG960 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC1020 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA1080 GGCCCTTTCG TCTTCAAGAA ttaaaaggat ctaggtgaag atcctttttg ataatctcatll40 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagatl200 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaal260 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaal320 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagttl380 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgttl440 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgatalSOO gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagcttl560 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccacl620 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggagalββO gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcgl740 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaalβOO aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacatl860 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagcl920 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcggal980 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaCAGA2040 TCTGtggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact2100 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac2160 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc2220 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gCCcagctgC2280 GATTCGAAct tctcgattcg aacttctgat agacttcgaa attaatacga ctcactatag2340 ggagaccaca acggtttccc tctagaaata attttgttta actttaagaa ggagatatac2400 atatgACCGT CCGCAAGAAC CAGGCGTCCC TGACCGCCGA GGAGAAGCGC CGCTTCGTCG2460 CCGCCCTGCT CGAACTCAAG CGCACCGGCC GCTACGACGC CTTCGTCACC ACGCACAACG2S20 CGTTCATCCT GGGCGACACC GACAACGGCG AGCGCACCGG CCACCGTTCG CCGTCCTTCC2580 TGCCCTGGCA CCGCAGATTT CTGCTGGAGT TCGAGCGGGC GCTCCAGTCG GTGGACGCGT2640 CGGTGGCGCT GCCGTACTGG GACTGGTCCG CCGACCGGTC CACCCGGTCC TCGCTGTGGG2700 CGCCGGACTT CCTCGGCGGC ACCGGGCGCA GCCGGGACGG CCAGGTGATG GACGGGCCGT2760 TCGCCGCGTC GGCCGGCAAC TGGCCGATCA ATGTGCGGGT GGACGGCCGT ACGTTCCTGC2820 GGCGGGCGCT CGGCGCGGGC GTGAGCGAAC TGCCCACGCG TGCCGAGGTC GACTCGGTGC2880 TGGCGATGGC GACGTACGAC ATGGCGCCCT GGAACAGCGG CTCCGACGGC TTCCGCAACC2940 ATCTCGAAGG GTGGCGCGGG GTCAATCTGC ACAACCGGGT GCATGTCTGG GTCGGCGGCC3000 AGATGGCGAC CGGGGTCTCC CCCAACGACC CGGTGTTCTG GCTGCACCAC GCCTACATCG3060 ACAAGCTGTG GGCCGAGTGG CAGCGGCGGC ACCCCTCGTC CCCGTATCTG CCGGGCGGCG3120 GCACGCCGAA CGTCGTCGAC CTCAACGAGA CGATGAAGCC GTGGAACGAC ACCACCCCGG3180 CGGCCCTGCT GGACCACACC CGGCACTACA CCTTCGACGT Ctggaattcc GCGGAACTCA3240 CCCGTCGTCG CGCGCTCGGC GCCGCAGCCG TCGTCGCCGC CGGTGTCCCG CTGGTCGCCC3300 TTCCCGCCGC CCGCGCGGAC GATCGGGGGC ACCACACCCC CGAGGTCCCC GGGAACCCGG3360 CCGCGTCCGG CGCCCCCGCC GCCTTCGACG AGATCTACAA GGGCCGCCGG ATACAGGGCC3420 GGACGGTCAC CGACGGCGGG GGCCACCACG GCGGCGGTCA CGGCGGTGAC GGTCACGGCG3 80 GCGGCCATCA CGGCGGCGGT TACGCCGTGT TCGTGGACGG CGTCGAACTG CATGTGATGC3540 GCAACGCCGA CGGCTCGTGG ATCAGCGTCG TCAGCCACTA CGAGCCGGTG GACACCCCGC3600 GCGCCGCGGC CCGCGCTGCG GTCGACGAGC TCCAGGGCGC CCGGCTCCTC CCCTTCCCCT3660 CCAACtgaCC TTCTCCCCCG CACTTTTGGA GCACCCGCAC atgACCGTCC GCAAGAACCA3720 GGCGTCCCTG ACCGCCGAGG AGAAGCGCCG CTTCGTCGCC GCCCTGCTCG AACTCAAGCG3780 CACCGGCCGC TACGACGCCT TCGTCACCAC GCACAACGCG TTCATCCTGG GCGACACCGA3840 CAACGGCGAG CGCACCGGCC ACCGTTCGCC GTCCTTCCTG CCCTGGCACC GCAGATTTCT3900 GCTGGAGTTC GAGCGGGCGC TCCAGTCGGT GGACGCGTCG GTGGCGCTGC CGTACTGGGA3960 CTGGTCCGCC GACCGGTCCA CCCGGTCCTC GCTGTGGGCG CCGGACTTCC TCGGCGGCAC4020 CGGGCGCAGC CGGGACGGCC AGGTGATGGA CGGGCCGTTC GCCGCGTCGG CCGGCAACTG4080 GCCGATCAAT GTGCGGGTGG ACGGCCGTAC GTTCCTGCGG CGGGCGCTCG GCGCGGGCGT4140 GAGCGAACTG CCCACGCGTG CCGAGgtcga cCTCAACGAG ACGATGAAGC CGTGGAACGA4200 CACCACCCCG GCGGCCCTGC TGGACCACAC CCGGCACTAC ACCTTCGACG TCtgaTCcaa4260 gc tATCGAT GATAAGCTGT CAAACATGAG AATT 4294 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 422 (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Thr Val Arg Lys Asn Gin Ala Ser Leu Thr Ala Glu Glu Lys 5 10 15 Arg Arg Phe Val Ala Ala Leu Leu Glu Leu Lys Arg Thr Gly Arg 20 25 30 Tyr Asp Ala Phe Val Thr Thr His Asn Ala Phe lie Leu Gly Asp 35 40 45 Thr Asp Asn Gly Glu Arg Thr Gly His Arg Ser Pro Ser Phe Leu 50 55 60 Pro Trp His Arg Arg Phe Leu Leu Glu Phe Glu Arg Ala Leu Gin 65 70 75 Ser Val Asp Ala Ser Val Ala Leu Pro Tyr Trp Asp Trp Ser Ala 80 85 90 Asp Arg Ser Thr Arg Ser Ser Leu Trp Ala Pro Asp Phe Leu Gly 95 100 105 Gly Thr Gly Arg Ser Arg Asp Gly Gin Val Met Asp Gly Pro Phe 110 115 120 Ala Ala Ser Ala Gly Asn Trp Pro lie Asn Val Arg Val Asp Gly 125 130 135 Arg Thr Phe Leu Arg Arg Ala Leu Gly Ala Gly Val Ser Glu Leu 140 145 150 Pro Thr Arg Ala Glu Val Asp Ser Val Leu Ala Met Ala Thr Tyr 155 160 165 Asp Met Ala Pro Trp Asn Ser Gly Ser Asp Gly Phe Arg Asn His 170 175 180 Leu Glu Gly Trp Arg Gly Val Asn Leu His Asn Arg Val His Val 185 190 195 Trp Val Gly Gly Gin Met Ala Thr Gly Val Ser Pro Asn Asp Pro 200 205 210 Val Phe Trp Leu His His Ala Tyr lie Asp Lys Leu Trp Ala Glu 215 220 225 Trp Gin Arg Arg His Pro Ser Ser Pro Tyr Leu Pro Gly Gly Gly 230 235 240 Thr Pro Asn Val Val Asp Leu Asn Glu Thr Met Lys Pro Trp Asn 245 250 255 Asp Thr Thr Pro Ala Ala Leu Leu Asp His Thr Arg His Tyr Thr 260 265 270 Phe Asp Val Trp Asn Ser Ala Glu Leu Thr Arg Arg Arg Ala Leu 275 280 285 Gly Ala Ala Ala Val Val Ala Ala Gly Val Pro Leu Val Ala Leu 290 295 300 Pro Ala Ala Arg Ala Asp Asp Arg Gly His His Thr Pro Glu Val 305 310 315 Pro Gly Asn Pro Ala Ala Ser Gly Ala Pro Ala Ala Phe Asp Glu 320 325 330 He Tyr Lys Gly Arg Arg He Gin Gly Arg Thr Val Thr Asp Gly 335 340 345 Gly Gly His His Gly Gly Gly His Gly Gly Asp Gly His Gly Gly 350 355 360 Gly His His Gly Gly Gly Tyr Ala Val Phe Val Asp Gly Val Glu 365 370 375 Leu His Val Met Arg Asn Ala Asp Gly Ser Trp He Ser Val Val 380 385 390 Ser His Tyr Glu Pro Val Asp Thr Pro Arg Ala Ala Ala Arg Ala 395 400 405 Ala Val Asp Glu Leu Gin Gly Ala Arg Leu Leu Pro Phe Pro Ser 410 415 420 Asn Gly (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CCAGGGCGCC CGGCTCCTCC CCTTCCCCTC CAACCA 36 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 42 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TCGAGGTCCC GCGGGCCGAG GAGGGGAAGG GGAGGTTGGT AT 42 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4009 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: circular (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: ( ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: AAATCAATCT AAAGTATATA TGAGTAAACT TGGTCTGACA GTTACCAATG CTTAATCAGT60 GAGGCACCTA TCTCAGCGAT CTGTCTATTT CGTTCATCCA TAGTTGCCTG ACTCCCCGTC120 GTGTAGATAA CTACGATACG GGAGGGCTTA CCATCTGGCC CAGTGCTGCA ATGATACCGC180 GAGACCCACG CTGACCGGCT CCAGATTTAT CAGCAATAAA CCAGCCAGCC GGAAGGGCCG240 AGCGCAGAAG TGGTCCTGCA ACTTTATCCG CCTCCATCCA GTCTATTAAT TGTTGCCGGG300 AAGCTAGAGT AAGTAGTTCG CCAGTTAATA GTTTGCGCAA CGTTGTTGCC ATTGCTACAG360 GCATCGTGGT GTCACGCTCG GCGTTTGGTA TGGCTTCATT CAGCTCCGGT TCCCAACGAT420 CAAGGCGAGT TACATGATCC CCCATGTTGT GCAAAAAAGC GGTTAGCTCC TTCGGTCCTC480 CGATCGTTGT CAGAAGTAAG TTGGCCGCAG TGTTATCACT CATGGTTATG GCAGCACTGC540 ATAATTCTCT TACTGTCATG CCATCCGTAA GATGCTTTTC TGTGACTGGT GAGTACTCAA600 CCAAGTATTT GGAAGATGCG CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA660 CCGCGCCACA TAGCAGAACT TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA720 AACTCTCAAG GATCTTACCG CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA780 ACTGATCTTC AGCATCTTTT ACTTTCACCA GCGTTTCTGG GTGAGCAAAA ACAGGAAGGC840 AAAATGCCGC AAAAAAGGGA ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC900 TTTTTCAATA TTATTGAAGC ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG960 AATGTATTTA GAAAAATAAA CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC1020 CTGACGTCTA AGAAACCATT ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA1080 GGCCCTTTCG TCTTCAAGAA ttaaaaggat ctaggtgaag atcctttttg ataatctcatll40 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagatl200 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaal260 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaal320 ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagttl380 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgttl440 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgatalSOO gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagcttl560 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccacl620 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggagal680 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcgl740 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaalβOO aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacatl860 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagcl920 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcggal980 agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaCAGA2040 TCTGtggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact2100 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac2160 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc2220 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gCCcagctgC2280 GATTCGAAct tctcgattcg aacttctgat agacttcgaa attaatacga ctcactatag2340 ggagaccaca acggtttccc TCTAGAaata attttgttta actttaagaa ggagatatac2400 atATGGCTAG AATTGCCatg GCGGAACTCA CCCGTCGTCG CGCGCTCGGC GCCGCAGCCG2460 TCGTCGCCGC CGGTGTCCCG CTGGTCGCCC TTCCCGCCGC CCGCGCGGAC GATCGGGGGC2520 ACCACACCCC CGAGGTCCCC GGGAACCCGG CCGCGTCCGG CGCCCCCGCC GCCTTCGACG2580 AGATCTACAA GGGCCGCCGG ATACAGGGCC GGACGGTCAC CGACGGCGGG GGCCACCACG2640 GCGGCGGTCA CGGCGGTGAC GGTCACGGCG GCGGCCATCA CGGCGGCGGT TACGCCGTGT2700 TCGTGGACGG CGTCGAACTG CATGTGATGC GCAACGCCGA CGGCTCGTGG ATCAGCGTCG2760 TCAGCCACTA CGAGCCGGTG GACACCCCGC GCGCCGCGGC CCGCGCTGCG GTCGACGAGC2820 TCCAGGGCGC CCGGCTCCTC CCCTTCCCCT CCAACcatAT GACCGTCCGC AAGAACCAGG2880 CGTCCCTGAC CGCCGAGGAG AAGCGCCGCT TCGTCGCCGC CCTGCTCGAA CTCAAGCGCA2940 CCGGCCGCTA CGACGCCTTC GTCACCACGC ACAACGCGTT CATCCTGGGC GACACCGACA3000 ACGGCGAGCG CACCGGCCAC CGTTCGCCGT CCTTCCTGCC CTGGCACCGC AGATTTCTGC3060 TGGAGTTCGA GCGGGCGCTC CAGTCGGTGG ACGCGTCGGT GGCGCTGCCG TACTGGGACT3120 GGTCCGCCGA CCGGTCCACC CGGTCCTCGC TGTGGGCGCC GGACTTCCTC GGCGGCACCG3180 GGCGCAGCCG GGACGGCCAG GTGATGGACG GGCCGTTCGC CGCGTCGGCC GGCAACTGGC3240 CGATCAATGT GCGGGTGGAC GGCCGTACGT TCCTGCGGCG GGCGCTCGGC GCGGGCGTGA3300 GCGAACTGCC CACGCGTGCC GAGGTCGACT CGGTGCTGGC GATGGCGACG TACGACATGG3360 CGCCCTGGAA CAGCGGCTCC GACGGCTTCC GCAACCATCT CGAAGGGTGG CGCGGGGTCA3420 ATCTGCACAA CCGGGTGCAT GTCTGGGTCG GCGGCCAGAT GGCGACCGGG GTCTCCCCCA3480 ACGACCCGGT GTTCTGGCTG CACCACGCCT ACATCGACAA GCTGTGGGCC GAGTGGCAGC3540 GGCGGCACCC CTCGTCCCCG TATCTGCCGG GCGGCGGCAC GCCGAACGTC GTCGACCTCA3600 ACGAGACGAT GAAGCCGTGG AACGACACCA CCCCGGCGGC CCTGCTGGAC CACACCCGGC3660 ACTACACCTT CGACGTCtga tcatcactga cgaatcgagg tcgaggaacc gagcgtccga3720 ggaacagagg cgcttatcgg ttggccgcga gattcctgtc gatcctctcg tgcagcgcga3780 ttccgaggga aacggaaacg ttgagagact cggtctggct catcatgggg atggaaaccg3840 aggcggaaga cgcctcctcg aacaggtcgg aaggcccacc cttttcgctg ccgaacagca3900 aggccagccg atccggattg tccccgagtt ccttcacgga aatgtcgcca tccgccttga3960 gcgtcatcag ATCaagcttA TCGATGATAA GCTGTCAAAC ATGAGAATT 4009 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 426 (B) TYPE: amino acid (C) STRANDEDNESS: (D) TOPOLOGY: circular (ii) MOLECULE TYPE: (A) DESCRIPTION: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: MET ALA ARG ILE ALA MET ALA GLU LEU THR ARG ARG ARG ALA LEU 5 10 15 GLY ALA ALA ALA VAL VAL ALA ALA GLY VAL PRO LEU VAL ALA LEU 20 25 30 PRO ALA ALA ARG ALA ASP ASP ARG GLY HIS HIS THR PRO GLU VAL 35 40 45 PRO GLY ASN PRO ALA ALA SER GLY ALA PRO ALA ALA PHE ASP GLU 50 55 60 ILE TYR LYS GLY ARG ARG ILE GLN GLY ARG THR VAL THR ASP GLY 65 70 75 GLY GLY HIS HIS GLY GLY GLY HIS GLY GLY ASP GLY HIS GLY GLY 80 85 90 GLY HIS HIS GLY GLY GLY TYR ALA VAL PHE VAL ASP GLY VAL GLU 95 100 105 LEU HIS VAL MET ARG ASN ALA ASP GLY SER TRP ILE SER VAL VAL 110 115 120 SER HIS TYR GLU PRO VAL ASP THR PRO ARG ALA ALA ALA ARG ALA 125 130 135 ALA VAL ASP GLU LEU GLN GLY ALA ARG LEU LEU PRO PHE PRO SER 140 145 150 ASN HIS MET THR VAL ARG LYS ASN GLN ALA SER LEU THR ALA GLU 155 160 165 GLU LYS ARG ARG PHE VAL ALA ALA LEU LEU GLU LEU LYS ARG THR 170 175 180 GLY ARG TYR ASP ALA PHE VAL THR THR HIS ASN ALA PHE ILE LEU 185 190 195 GLY ASP THR ASP ASN GLY GLU ARG THR GLY HIS ARG SER PRO SER 200 205 210 PHE LEU PRO TRP HIS ARG ARG PHE LEU LEU GLU PHE GLU ARG ALA 215 215 220 LEU GLN SER VAL ASP ALA SER VAL ALA LEU PRO TYR TRP ASP TRP 225 230 235 SER ALA ASP ARG SER THR ARG SER SER LEU TRP ALA PRO ASP PHE 240 245 250 LEU GLY GLY THR GLY ARG SER ARG ASP GLY GLN VAL MET ASP GLY 255 260 265 PRO PHE ALA ALA SER ALA GLY ASN TRP PRO ILE ASN VAL ARG VAL 270 275 280 ASP GLY ARG THR PHE LEU ARG ARG ALA LEU GLY ALA GLY VAL SER 285 290 295 GLU LEU PRO THR ARG ALA GLU VAL ASP SER VAL LEU ALA MET ALA 300 305 310 THR TYR ASP MET ALA PRO TRP ASN SER GLY SER ASP GLY PHE ARG 315 320 325 ASN HIS LEU GLU GLY TRP ARG GLY VAL ASN LEU HIS ASN ARG VAL 330 335 340 HIS VAL TRP VAL GLY GLY GLN MET ALA THR GLY VAL SER PRO ASN 345 350 355 ASP PRO VAL PHE TRP LEU HIS HIS ALA TYR ILE ASP LYS LEU TRP 360 370 375 ALA GLU TRP GLN ARG ARG HIS PRO SER SER PRO TYR LEU PRO GLY 380 385 390 GLY GLY THR PRO ASN VAL VAL ASP LEU ASN GLU THR MET LYS PRO 395 400 405 TRP ASN ASP THR THR PRO ALA ALA LEU LEU ASP HIS THR ARG HIS 410 415 420 TYR THR PHE ASP VAL GLY 425 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1442 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 8 : ctcgagccAT GGCTTCCTCA GTTCTTTCCT CTGCAGCAGT TGCCACCCGC AGCAATGTTG60 CTCAAGCTAA CATGGTTGCA CCTTTCACTG GCCTTAAGTC AGCTGCCTCA TTCCCTGTTT120 CAAGGAAGCA AAACCTTGAC ATCACTTCCA TTGCCAGCAA CGGCGGAAGA GTGCAATGCAl 80 TGCCGGAACT CACCCGTCGT CGCGCGCTCG GCGCCGCAGC CGTCGTCGCC GCCGGTGTCC240 CGCTGGTCGC CCTTCCCGCC GCCCGCGCGG ACGATCGGGG GCACCACACC CCCGAGGTCC300 CCGGGAACCC GGCCGCGTCC GGCGCCCCCG CCGCCTTCGA CGAGATCTAC AAGGGCCGCC360 GGATACAGGG CCGGACGGTC ACCGACGGCG GGGGCCACCA CGGCGGCGGT CACGGCGGTG420 ACGGTCACGG CGGCGGCCAT CACGGCGGCG GTTACGCCGT GTTCGTGGAC GGCGTCGAAC480 TGCATGTGAT GCGCAACGCC GACGGCTCGT GGATCAGCGT CGTCAGCCAC TACGAGCCGG540 TGGACACCCC GCGCGCCGCG GCCCGCGCTG CGGTCGACGA GCTCCAGGGC GCCCGGCTCC600 TCCCCTTCCC CTCCAACCAT ATGACCGTCC GCAAGAACCA GGCGTCCCTG ACCGCCGAGG660 AGAAGCGCCG CTTCGTCGCC GCCCTGCTCG AACTCAAGCG CACCGGCCGC TACGACGCCT720 TCGTCACCAC GCACAACGCG TTCATCCTGG GCGACACCGA CAACGGCGAG CGCACCGGCC780 ACCGTTCGCC GTCCTTCCTG CCCTGGCACC GCAGATTTCT GCTGGAGTTC GAGCGGGCGC840 TCCAGTCGGT GGACGCGTCG GTGGCGCTGC CGTACTGGGA CTGGTCCGCC GACCGGTCCA900 CCCGGTCCTC GCTGTGGGCG CCGGACTTCC TCGGCGGCAC CGGGCGCAGC CGGGACGGCC960 AGGTGATGGA CGGGCCGTTC GCCGCGTCGG CCGGCAACTG GCCGATCAAT GTGCGGGTGG1020 ACGGCCGTAC GTTCCTGCGG CGGGCGCTCG GCGCGGGCGT GAGCGAACTG CCCACGCGTG1080 CCCAGGTCGA CTCGGTGCTG GCGATGGCGA CGTACGACAT GGCGCCCTGG AACAGCGGCT1140 CCGACGGCTT CCGCAACCAT CTCGAAGGGT GGCGCGGGGT CAATCTGCAC AACCGGGTGC1200 ATGTCTGGGT CGGCGGCCAG ATGGCGACCG GGGTCTCCCC CAACGACCCG GTGTTCTGGC1260 TGCACCACGC CTACATCGAC AAGCTGTGGG CCGAGTGGCA GCGGCGGCAC CCCTCGTCCC1320 CGTATCTGCC GGGCGGCGGC ACGCCGAACG TCGTCGACCT CAACGAGACG ATGAAGCCGT1380 GGAACCACAC CACCCCGGCG GCCCTGCTGG ACCACACCCG GCACTACACC TTCGACGTCT1440 GA 1442 (2) INFORMATION FOR SEQ ID NO: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 478 (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: (A) DESCRIPTION: DNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: (vii) IMMEDIATE SOURCE: (B) CLONE: (ix) FEATURE: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Ala Ser Ser Val Leu Ser Ser Ala Ala Val Ala Thr Arg Ser 5 10 15 Asn Val Ala Gin Ala Asn Met Val Ala Pro Phe Thr Gly Leu Lys 20 25 30 Ser Ala Ala Ser Phe Pro Val Ser Arg Lys Gin Asn Leu Asp lie 35 40 45 Thr Ser lie Ala Ser Asn Gly Gly Arg Val Gin Cys Met Pro Glu 50 55 60 Leu Thr Arg Arg Arg Ala Leu Gly Ala Ala Ala Val Val Ala Ala 65 70 75 Gly Val Pro Leu Val Ala Leu Pro Ala Ala Arg Ala Asp Asp Arg 80 85 90 Gly His His Thr Pro Glu Val Pro Gly Asn Pro Ala Ala Ser Gly 95 100 105 Ala Pro Ala Ala Phe Asp Glu lie Tyr Lys Gly Arg Arg lie Gin 110 115 120 Gly Arg Thr Val Thr Asp Gly Gly Gly Asp His Gly Gly Gly His 125 130 135 Gly Gly Asp Gly His Gly Gly Gly His His Gly Gly Gly Tyr Ala 140 145 150 Val Phe Val Asp Gly Val Glu Leu His Val Met Arg Asn Ala Asp 155 160 165 Gly Ser Trp He Ser Val Val Ser His Tyr Glu Pro Val Asp Thr 170 175 180 Pro Arg Ala Ala Ala Arg Ala Ala Val Asp Glu Leu Gin Gly Ala 185 190 195 Arg Leu Leu Pro Phe Pro Ser Asn His Met Thr Val Arg Lys Asn 200 205 210 Gin Ala Ser Leu Thr Ala Glu Glu Lys Arg Arg Phe Val Ala Ala 215 215 220 Leu Leu Glu Leu Lys Arg Thr Gly Arg Tyr Asp Ala Phe Val Thr 225 230 235 Thr His Asn Ala Phe He Leu Gly Asp Thr Asp Asn Gly Glu Arg 240 245 250 Thr Gly His Arg Ser Pro Ser Phe Leu Pro Trp His Arg Arg Phe 255 260 265 Leu Leu Glu Phe Glu Arg Ala Leu Gin Ser Val Asp Ala Ser Val 270 275 280 Ala Leu Pro Tyr Trp Asp Trp Ser Ala Asp Arg Ser Thr Arg Ser 285 290 295 Ser Leu Trp Ala Pro Asp Phe Leu Gly Gly Thr Gly Arg Ser Arg 300 305 310 Asp Gly Gin Val Met Asp Gly Pro Phe Ala Ala Ser Ala Gly Asn 315 320 325 Trp Pro He Asn Val Arg Val Asp Gly Arg Thr Phe Leu Arg Arg 330 335 340 Ala Leu Gly Ala Gly Val Ser Glu Leu Pro Thr Arg Ala Glu Val 345 350 355 Asp Ser Val Leu Ala Met Ala Thr Tyr Asp Met Ala Pro Trp Asn 360 370 375 Ser Gly Ser Asp Gly Phe Arg Asn His Leu Glu Gly Trp Arg Gly 380 385 390 Val Asn Leu His Asn Arg Val His Val Trp Val Gly Gly Gin Met 395 400 405 Ala Thr Gly Val Ser Pro Asn Asp Pro Val Phe Trp Leu His His 410 415 420 Ala Tyr He Asp Lys Leu Trp Ala Glu Trp Gin Arg Arg His Pro 425 430 435 Ser Ser Pro Tyr Leu Pro Gly Gly Gly Thr Pro Asn Val Val Asp 440 445 450 Leu Asn Glu Thr Met Lys Pro Trp Asn Asp Thr Thr Pro Ala Ala 455 460 465 Leu Leu Asp His Thr Arg His Tyr Thr Phe Asp Val Gly 470 475 International Application No: PCT/ / MICROORGANISMS Optional Sheet in connection with the microorganism referred to on page , lines of the description ' A. IDENTIFICATION OF DEPOSIT ' Further deposits are identified on an additional sheet Name of depositary institution ' American Type Culture Collection Address of depositary institution (including postal code and country) ' 12301 Parklawn Drive Rockville, MD 20852 US Date of deposit ' December 28, 1993 Accession Number ' 75632 B. ADDITIONAL INDICATIONS ' fleave blank if not applicable) . Thu information a continued on a separate attached sheet C. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE ' or*. D. SEPARATE FURNISHING OF INDICATIONS ' (leave blank if not applicable) The indications listed below will be submitted to the International Bureau later * (Specify the general nature of the indications e.g., "Accession Number of Deposit") E. D This sheet was received with the International applicanon when filed (to be checked by the receiving Office) (Authorized Officer) □ The date of receipt (from the applicant) by the International Bureau ' was (Authorized Officer) Form PCf/f-O/134 (January 1981 )

Claims: WHAT IS CLAIMED IS: 1. A nucleic acid sequence encoding a fusion enzyme comprising a nucleic acid sequence encoding for a tyrosinase and a nucleic acid sequence encoding for a tyrosinase activator protein. 2. A nucleic acid sequence encoding a fusion enzyme according to claim 1 wherein the tyrosinase is derived from a pro aryote. 3. A nucleic acid sequence encoding a fusion enzyme according to claim 2 wherein the tyrosinase is derived from Streptomyces- 4. A nucleic acid sequence encoding a fusion enzyme according to claim l wherein the activator protein is selected from the group consisting of ORF438 and URF402. 5. A nucleic acid sequence encoding a fusion enzyme according to claim l wherein the activator protein is ORF438 and the tyrosinase is derived from S. antibioticus. 6. - A nucleic acid sequence encoding a fusion enzyme according to claim 1 wherein the activator protein sequence is positioned 5' relative to the tyrosinase sequence. 7. A nucleic acid sequence encoding a fusion enzyme according to claim 1 wherein the tyrosinase sequence is positioned 5' relative to the the activator protein sequence. 8. A nucleic acid sequence encoding a fusion enzyme according to claim 6 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 9. A nucleic acid sequence encoding a fusion enzyme according to claim 6 wherein the tyrosinase is derived from a prokaryote. 10. A nucleic acid sequence encoding a fusion enzyme according to claim 9 wherein the tyrosinase is derived from Streptomvces. 11. A nucleic acid sequence encoding a fusion enzyme according to claim 1 which further comprises a linker sequence positioned between the nucleic acid sequences encoding the activator protein and the tyrosinase. 12. A nucleic acid sequence encoding a fusion enzyme according to claim 11 wherein linker sequence encodes for a single amino acid. 13. A nucleic acid sequence encoding a fusion enzyme according to claim 12 wherein linker sequence encodes for a histidine residue. 1 . A nucleic acid sequence encoding a fusion enzyme according to claim 12 wherein linker sequence encodes for a repeating Pro-Thr amino acid sequence. 15. A nucleic acid sequence encoding a fusion enzyme according to claim 11 wherein the nucleic acid sequence encoding the activator protein is positioned 5' relative to the nucleic acid sequence encoding the tyrosinase. 16. A nucleic acid sequence encoding a fusion enzyme according to claim 15 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 17. A nucleic acid sequence encoding a fusion enzyme according to claim 15 wherein the tyrosinase is derived fro a prokaryote. 18. A nucleic acid sequence encoding a fusion enzyme according to claim 17 wherein the tyrosinase is derived fro Streptomyces. 19. A vector for transforming an organism, said vector comprising: a nucleic acid sequence encoding for a fusion enzyme, said fusion enzyme comprising an amino acid sequence for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein; and a promoter sequence that regulates the transcription of fusion enzyme. 20. A vector according to claim 19 wherein the activator protein sequence is positioned 5 1 relative to the tyrosinase sequence. 21. A vector according to claim 19 wherein the tyrosinase sequence is positioned 5* relative to the the activator protein sequence. 22. A vector according to claim 19 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 23. A vector according to claim 19 wherein the tyrosinase is derived from a prokaryote. 24. A vector according to claim 23 wherein the tyrosinase is derived from Streptomyces. 25. A vector according to claim 19 wherein the activato protein is ORF438 and the tyrosinase is S. antibioticus. 26. A vector according to claim 19 which further comprises a linker sequence positioned between the nucleic acid sequences encoding the activator protein and the tyrosinase. 27. A vector according to claim 26 wherein linker sequence encodes for a single amino acid. 28. A vector according to claim 27 wherein linker sequence encodes for a histidine residue. 29. A vector according to claim 26 wherein linker sequence encodes for a repeating Pro-Thr amino acid sequence. 30. A vector according to claim 19 wherein the activato protein is selected from the group consisting of ORF438 an URF402. 31. A vector according to claim 19 wherein the tyrosinase is derived from S. antibioticus. 32. A fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein. 33. A fusion enzyme according to claim 32 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 34. A fusion enzyme according to claim 32 wherein the tyrosinase is derived from a prokaryote. 35. A fusion enzyme according to claim 34 wherein the tyrosinase is derived from Streptomyces. 36. A fusion enzyme according to claim 32 wherein the activator protein is ORF438 and the tyrosinase is derived from S. antibioticus. 37. A fusion enzyme according to claim 32 which further comprises a linker positioned between the amino acid sequences of the activator protein and the tyrosinase. 38. A fusion enzyme according to claim 37 wherein the linker sequence comprises a single amino acid. 39. A fusion enzyme according to claim 38 wherein the linker sequence comprises a histidine residue. 40. A fusion enzyme according to claim 37 wherein the linker sequence comprises a repeating Pro-Thr amino acid sequence. 41. A fusion enzyme according to claim 32 wherein the activator protein sequence is positioned on the N-terminus end of the tyrosinase sequence. 42. A fusion enzyme according to claim 41 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 43. A fusion enzyme according to claim 41 wherein the tyrosinase is derived from a prokaryote. 44. A fusion enzyme according to claim 43 wherein the tyrosinase is derived from Streptomyces. 45. A fusion enzyme according to claim 34 wherein the activator protein sequence is positioned on the C-terminus end of the tyrosinase sequence. 46. A fusion enzyme according to claim 42 wherein the activator protein is selected from the group consisting of ORF438 and URF402. 47. A fusion enzyme according to claim 42 wherein the tyrosinase is derived from a prokaryote. 48. A fusion enzyme according to claim 47 wherein the tyrosinase is derived from Streptomyces. 49. A melanin wherein said melanin is produced by a transformed organism expressing a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein. 50. A melanin according to claim 49 wherein the transformed organism is selected from the group consisting of bacteria, yeast fungi, plants and animals. 51. An in vitro method of producing a melanin comprising the steps of contacting a fusion enzyme comprising an amino acid sequence for tyrosinase and an amino acid sequence for a tyrosinase activator protein and a reaction substrate selected from the group consisting of L-tyrosine, X/L-tyrosine, L-tyroεine/X and X/L-tyrosine/X where X is a single amino acid, a dipeptide or an oligopeptide bound to L-tyrosine, under suitable reaction conditions, to form melanin. 52. An organism which has been transformed with a vecto comprising a nucleic acid sequence encoding for a fusion enzyme sai fusion enzyme comprising an amino acid sequence for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein; and a promoter sequence that regulates the transcription of fusion enzyme. 53. An organism according to claim 45 wherein the transformed organism is selected from the group consisting of bacteria, yeast fungi, plants and animals. 54. A method of producing melanins, comprising the step of growing an organism which has been transformed with a vector comprising a nucleic acid sequence encoding for a fusion enzyme, said fusion enzyme comprising an amino acid sequence for a tyrosinase and an amino acid sequence encoding for a tyrosinase activator protein; and a promoter sequence that regulates the transcription of fusion enzyme. 55. A method according to claim 54 comprising the further step of isolating the melanin produced.

Inventors: DELLA-CIOPPA GUY, KUMAGAI MONTO H, DELLA-CIOPPA, GUY, KUMAGAI, MONTO, H.
Applicants: BIOSOURCE TECH INC, BIOSOURCE TECHNOLOGIES INCORPORATED
Application year: 1994
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N1, C12N9, C12N15, C12P17, C12R1